Scanning electron microscopy (SEM) of blood clots.

<p>Representative image of SEM analysis of blood clots formed during thrombin generation measurements. Sequential enlargements of the fibrin network with entrapped red blood cells are depicted. <b>(A)</b> Zebrafish blood clot. <b>(B)</b> Human blood clot without added tissue factor (TF). <b>(C)</b> Human blood clot with addition of a high dose (100 pM) of TF.</p

Inhibition of thrombin generation in zebrafish by inhibition of platelet aggregation.

<p>Zebrafish were treated with 30 μg/g aspirin or vehicle for 30 min <b>(A-B)</b> or were allowed to swim in aspirin-supplemented water (250 mg/ml) for 24 hours <b>(C-D)</b>, followed by thrombin generation measurements. <b>(A,C)</b> Thrombin generation curves are shown. <b>(B,D)</b> Results are expressed as the percentage inhibition compared to a control group of vehicle-treated fish (n = 5). The percentage CV is indicated for all parameters. *Mann-Whitney U-test as compared to controls.</p

Thrombin generation curves and parameters in zebrafish, effect of temperature and comparison with humans.

<p>Thrombin generation was measured in zebrafish at 28°C and 37°C and compared to thrombin generation in humans at 37°C with low (no tissue factor (TF) added) or high (100 pM) TF. <b>(A)</b> Thrombin generation curves are shown (n = 8 or 9 for fish, n = 3 for humans). <b>(B)</b> Mean thrombin generation parameters and their CV are calculated. Statistical differences as compared to the zebrafish group measured at 37°C are indicated (*p<0.05, **p<0.01, ***p<0.0001, 1-way ANOVA). <b>(C-D)</b> Thrombin generation was measured in 6 independent experiments, in a total of 34 zebrafish. <b>(C)</b> Thrombin generation curves are shown. Each color represents an independent experiment. <b>(D)</b> Mean thrombin generation parameters and their CV are calculated, as well as the CV between the means of all 6 experiments (interexp. CV).</p

Inhibition of thrombin generation in zebrafish by thrombin or FXa inhibition.

<p>Zebrafish were treated with melagatran <b>(A)</b> or rivaroxaban <b>(B)</b> at the indicated doses for 30 min, followed by thrombin generation measurements. <b>(C-D)</b> Results of two independent experiments are expressed as the percentage inhibition compared to a control group of vehicle-treated fish (n = 8). The percentage CV is indicated for all parameters. Statistical differences between treatment and control groups were performed with *Mann-Whitney U-test or ^$1-way ANOVA. (ns = not significant).</p

Standardization and reference ranges for whole blood platelet function measurements using a flow cytometric platelet activation test

<div><p>Introduction</p><p>Platelet function testing with flow cytometry has additional value to existing platelet function testing for diagnosing bleeding disorders, monitoring anti-platelet therapy, transfusion medicine and prediction of thrombosis. The major challenge is to use this technique as a diagnostic test. The aim of this study is to standardize preparation, optimization and validation of the test kit and to determine reference values in a population of 129 healthy individuals.</p><p>Methods</p><p>Platelet function tests with 3 agonists and antibodies against P-selectin, activated αIIbβ3 and glycoprotein Ib (GPIb), were prepared and stored at -20°C until used. Diluted whole blood was added and platelet activation was quantified by the density of activation markers, using flow cytometry. Anti-mouse Ig κ particles were included to validate stability of the test and to standardize results. Reference intervals were determined.</p><p>Results</p><p>Blood stored at room temperature (RT) for up to 4h after blood donation and preheated/tested at 37°C resulted in stable results (%CV<10%), in contrast to measuring at RT. The intra-assay %CV was <5%. Incubation of anti-mouse Ig κ particles with antibodies stored for up to 12 months proved to give a stable fluorescence. The inter-individual variation measured in the 129 individuals varied between 23% and 37% for P-selectin expression and αIIbβ3 activation, respectively.</p><p>Conclusions</p><p>The current study contributes to the translation of flow cytometry based platelet function testing from a scientific tool to a diagnostic test. Platelet function measurements, using prepared and stored platelet activation kits, are reproducible if executed at 37°C. The reference ranges can be validated in clinical laboratories and ongoing studies are investigating if reduced platelet reactivity in patients with bleeding complications can be detected.</p></div

Assay variation when blood is stored at RT up till 240 min followed by 10 min preincubation at 37°C prior to performing the platelet function test.

<p>Assay variation when blood is stored at RT up till 240 min followed by 10 min preincubation at 37°C prior to performing the platelet function test.</p

Effect of temperature on platelet function testing by flow cytometry.

<p>Immediately after blood donation, blood was stored at RT or at 37°C. At each time point (X-axis), platelet activation in response to TRAP (panel a), CRP (panel b) or ADP (panel c) was tested. For the 37°C condition, blood was immediately placed at 37° and platelet function tests were performed at 37°C. For the RT condition, blood was kept at RT and the tests were performed at RT. For the RT/37°C condition, blood was kept at RT and 10 min before performing the platelet function test, blood was placed at 37°C and tests were performed at 37°C. The median fluorescence intensity (MFI) of αIIbβ3 receptor activation (left panels) and P-selectin expression (right panels) was recorded and expressed as the percentage compared to the MFI of the 37°C condition measured after 30 min at 37°C. Data are presented as mean ± SD (n = 3 to 5).</p

Intra assay variability of the platelet function test and beads.

<p>Intra assay variability of the platelet function test and beads.</p

Inter-individual variation and reference intervals of the platelet function test.

<p>Inter-individual variation and reference intervals of the platelet function test.</p

Accessory cells (ACs) of immunised IFN-γR KO mice are required for their defective Tactivity

<p><b>Copyright information:</b></p><p>Taken from "Defective CD4CD25regulatory T cell functioning in collagen-induced arthritis: an important factor in pathogenesis, counter-regulated by endogenous IFN-γ"</p><p>Arthritis Research & Therapy 2005;7(2):R402-R415.</p><p>Published online 28 Jan 2005</p><p>PMCID:PMC1065335.</p><p>Copyright © 2005 Kelchtermans et al.; licensee BioMed Central Ltd.</p> Tcells, Tcells and ACs were isolated from lymph nodes and spleen of IFN-γR KO and wild-type DBA/1 mice 21 days after immunisation with collagen type II in complete Freund's adjuvant. Mixing experiments were performed as indicated. In each set, 5 × 10CD4CD25Tcells were incubated with anti-CD3 antibody in the presence of ACs and the indicated number of CD4CD25Tcells. The percentage inhibition of the proliferation of Tcells (CD4CD25) by increasing numbers of CD4CD25Tcells is shown. The results are representative of two independent experiments